1. Field of the Invention
The present invention relates to a use of protein nanoparticle-based hydrogel capable of highly sensitive and simultaneous multi-detection of disease markers by using a hydrogel within which protein nanoparticles presenting multiple copies of disease marker detection probes are immobilized.
2. Discussion of Related Art
In protein detecting technology based on specific protein-protein interactions, for example, antigen-antibody, it is important to activate a protein probe and maintain a specific binding capacity to a target molecule. Many conventional methods for attaching proteins to solid substrate surfaces of various protein chips are carried out by simple adsorption/spread or based on immobilization through covalent bond between primary amine groups on proteins. However, typically, a protein is randomly attached to a substrate surface and a structure of the protein is easily modified, so that activity of the protein is inhibited and the protein cannot be bound to a material on the surface, resulting in low efficiency of specific binding. Further, if a protein probe is immobilized on a substrate surface by simple adsorption, the protein probe may be washed away by intensive washing conditions during a detection process, or may be transferred to another molecule having a higher affinity to the substrate surface, and particularly, it may be difficult to quantitatively control the protein probe immobilized on the substrate surface of protein chip and maintain activity of the protein probe [Kusnezow, W. Hoheisel, J. D. J. Mol. Recognit. 16, 165-176 (2003); Park, J. S. et al. Nat. Nanotechnol. 4, 259-264 (2009); Kingsmore, S. F. Nat Rev Drug Discov. 5(4), 310-20 (2006); Ellington, A. A., Kullo, I. J., Bailey, K. R. & Klee, G. G. Clin. Chem. 56(2), 186-193 (2010)].
Unlike conventional organic and inorganic nanoparticles (metal nanoparticles) which are artificially synthesized, protein nanoparticles as nanomaterials synthesized by self-assembly in a cell of a living organism can secure uniform particle size distribution and stability and can be easily mass-produced in a cell of a microorganism. Further, the protein nanoparticles can be developed to have various characteristics/functions by genetically engineered surface modification. In particular, when a disease marker detecting peptide or protein (disease marker detection probe) is represented on a surface, the protein nanoparticles can secure uniform orientation, high-density integration, and structural stability. Thus, the protein nanoparticles have been used as a material of a probe for a highly sensitive diagnostic system [Park, J. S. et al. Nat. Nanotechnol. 4, 259-264 (2009); Seo, H. S. et al. Adv. Funct. Mater. 20, 4055-4061 (2010); Lee, J. H. et al. Adv. Funct. Mater. 20, 2004-2009 (2010); Lee, S. H. et al. The FASEB J. 21, 1324-1334 (2007)].
A hydrogel has a three-dimensional porous structure and can maintain a uniform content of moisture therein, and, thus, the hydrogel has been widely used for analyzing and utilizing proteins. In particular, when the hydrogel forms a polymer through a certain coupling reaction, the hydrogel can form a covalent bond with a material having a specific residue. Thus, the hydrogel has been widely used for immobilizing a functional material. If a protein such as an enzyme is immobilized within a hydrogel, it is possible to maintain activity of the enzyme for a long time [Nolan, J. P. TRENDS in Biotechnology. 20, 9-12 (2002)]. However, if only a hydrogel is used as an enzyme support, the hydrogel is swollen by moisture and enzymes are spread out of the hydrogel, and thus stability over time is sharply decreased [Basri, M. et al. J. Appl. Polym. Sci. 82, 1404-1409 (2001)]. Therefore, technology for maintaining activity of a protein enzyme or a protein probe for a long time while immobilizing it in a moistened hydrogel is needed.
Accordingly, in the present invention, among incurable diseases, Sjögren's syndrome and acquired immune deficiency syndrome which cannot be clinically diagnosed from symptoms only are selected as model diseases, protein nanoparticle presenting multiple copies of detecting probes specific to the two diseases on a surface of the protein nanoparticle is synthesized, and a three-dimensional diagnostic sensor system having maximized surface area and stability is developed by fusing the protein nanoparticles with a three-dimensional porous hydrogel so as to construct a practical diagnostic system capable of highly sensitive and simultaneous multi-detection.